Denitrification with methane as external carbon source
Introduction
Biological denitrification of nitrate-contaminated waters poor in organic content requires an external electron donor. Methane is a potentially inexpensive, widely available electron donor for denitrification of drinking water (in situ or at a treatment plant), wastewater or landfill leachate. In the latter two cases (wastewater and landfill leachate) methane would be especially suitable since it is generated onsite due to the anaerobic digestion of sludge in wastewater treatment plants and degradation of organic waste in landfills. Based on stoichiometric Eq. (1), which is thermodynamically favorable under standard conditions (ΔGfo=−767 kJ mol−1 CH4), nitrate is reduced to dinitrogen and methane is oxidized to carbon dioxide.
Since the 1970s there have been several investigations of denitrification with methane as external carbon source. Harremoes and Henze Christensen (1971) observed nitrate removal in a flask supplied with nitrified domestic wastewater under a pure methane headspace. Sediment from a severely eutrophic lake was used as inoculum. No nitrate removal was observed in an identical flask with nitrogen gas in the headspace. These results indicated that denitrification with methane is possible, though methane consumption and the fate of the removed nitrate were not investigated in this study (Harremoes and Henze Christensen, 1971). Davies (1973) claimed to have isolated bacteria capable of using methane as well as other carbon compounds as electron donors in denitrification. However, the liquid medium used contained a vitamin solution based on ethanol and it was concluded that nonmethanotrophic bacteria had probably been enriched in these experiments (Mason, 1977). Sollo et al. (1976) compared denitrification with methanol and methane as electron donors in packed columns and fluidized beds. They found denitrification with methanol was significantly higher than with methane, 4.6 mg N l−1 h−1 compared to 0.7 mg N l−1 h−1, in gravel-packed columns. A somewhat higher methane denitrification rate was obtained in an activated carbon fluidized bed (1.2 mg N l−1 h−1), but half of that rate (0.6 mg N l−1 h−1) could be attributed to endogenous decay of organic matter in the reactor rather than methane oxidation (Sollo et al., 1976). Similarly, Mason (1977) observed low denitrification rates in biodisc units supplied with methane gas. Due to the low rates obtained, it was concluded that denitrification according to Eq. (1) was unlikely to occur (Mason, 1977).
Currently, there is no known methanotroph that can carry out denitrification (Knowles, 2005). However, most studies have until now focused on denitrification with methane under aerobic conditions. In the presence of oxygen, methane is oxidized by aerobic methanotrophs releasing soluble organics that are used by coexisting denitrifiers. This process was first demonstrated by Rhee and Fuhs (1978) and later Meschner and Hamer (1985). Recent studies have shown that denitrification with methane is also possible under anaerobic conditions (Islas-Lima et al., 2004) and is performed by a microbial consortium consisting of archaea and bacteria (Raghoebarsing et al., 2006).
In reviewing denitrification with methane as external carbon source the different microbial processes responsible for methane oxidation and nitrate reduction must be clearly distinguished. Methane may be oxidized either aerobically or anaerobically in two completely different microbial processes. These two processes are accomplished by very different microbial communities (described in Section 2 of this review). Both processes, however, may be coupled to denitrification (i.e. the dissimilative reduction of nitrate to dinitrogen). When describing anaerobic methane oxidation, it is relevant to include sulfate reduction since sulfate is the most extensively studied electron acceptor for this process. This review uses the following notations for these microbial processes:
AME: Aerobic methane oxidation.
ANME: Anaerobic methane oxidation.
AME-D: Aerobic methane oxidation coupled to denitrification.
ANME-D: Anaerobic methane oxidation coupled to denitrification.
ANME-SR: Anaerobic methane oxidation coupled to sulfate reduction.
A bioreactor configuration, aerobic or anaerobic, in which the denitrification is dependent on the oxidation of methane, is referred to as a ME-D reactor. To specify aerobic or anaerobic conditions the notations AME-D reactor or ANME-D reactor are used.
Furthermore, some studies (Lee et al., 2001; Khin and Annachhatre, 2004a) have been carried out on simultaneous nitrification and denitrification (SND) using methane as external carbon source. This process is denoted:
ME-SND: Methane oxidation coupled to simultaneous nitrification and denitrification.
Besides denitrification, assimilation of nitrate by growing microorganisms also contributes to the total nitrate removal from a ME-D reactor. Particularly nitrate assimilation by aerobic methanotrophs may have contributed to nitrate removal in many of the studies discussed in this review. The term AME-D, however, does not include nitrate reduced for assimilatory purposes.
The aim of this paper is to review the literature on denitrification with methane as external carbon source and to identify problems preventing the practical application of this process. Although the ANME-D and ME-SND processes are described, focus is placed on AME-D since up until now most research has been carried out in this area.
Section snippets
Aerobic methane oxidizers
Aerobic methane oxidation (AME) is carried out by methanotrophs, which are strictly aerobic bacteria able to use methane as their sole source of carbon and energy. They are ubiquitous in nature and have been found in samples taken from all kinds of environments including swamps, soils, rivers, oceans, ponds, sewage sludge, etc. Methanotrophs are a subgroup of methylotrophs, which are bacteria using one-carbon compounds more reduced than formic acid as carbon and energy source and assimilate
Mechanism of AME-D
Denitrification with methane under aerobic conditions is carried out by a microbial consortium consisting of aerobic methanotrophs oxidizing methane and denitrifiers using organic compounds released by the methanotrophs as the electron donor (see Fig. 2).
An association between methanotrophic and denitrifying bacteria surviving on methane as the sole carbon source and accomplishing denitrification was first demonstrated by Rhee and Fuhs (1978). Bacteria were isolated from enrichment cultures of
Methane oxidation coupled to SND (ME-SND)
The idea of SND is that nitrification and denitrification occur in the same reactor under the same overall operating conditions. Nitrifiers require oxygen and are generally autotrophic whereas denitrification is inhibited by oxygen and generally requires an organic carbon source. A parallel can be drawn to the AME-D process, which assumes the same kind of contradictory coexistence between aerobic methanotrophs and anaerobic denitrifiers. This has led researchers to examine the possibility of
Anaerobic methane oxidation coupled to denitrification (ANME-D)
ANME-SR has been observed in marine sediments by an association of methane-oxidizing archaea and sulfate-reducing bacteria (SRB) (Valentine and Reeburgh, 2000; Hinrichs and Boetius, 2002; Valentine, 2002; Strous and Jetten, 2004). Recently, Raghoebarsing et al. (2006) demonstrated that ANME may also be coupled to denitrification. In this case, nitrate instead of sulfate, serves as the terminal oxidant. Anaerobic sediments from a freshwater canal subjected to agricultural runoff were used as
Comparison of denitrification rates
A compilation of denitrification rates obtained by different researchers on ME-D is shown in Table 1. Rates on a per biomass basis are available from suspended growth systems and can be compared to typical values from wastewater treatment plants (see Metcalf & Eddy et al., 1991). It should be noted that the nitrate removal rates from AME-D reactors do not clearly distinguish between denitrification and assimilation, whereas the rates from ANME-D systems (Raghoebarsing et al., 2006; Islas-Lima
Basis for evaluation
Two theoretical case studies are carried out to investigate the feasibility of applying ME-D reactors for denitrification of wastewater (case 1, Section 7.2) and landfill leachate (case 2, Section 7.3). The studies examine whether the methane gas produced on-site is sufficient for denitrification. Furthermore, it is investigated how cost competitive the ME-D processes are compared to denitrification with methanol as external carbon source (Section 7.4). Finally, problems associated with the
Conclusions
Several studies have shown methane could be used as a carbon source for biological denitrification. Most studied is the AME-D process, in which denitrification occurs by an association of methanotrophic and denitrifying organisms, where the aerobic methanotrophs oxidize methane and release organic compounds that are used by coexisting denitrifiers. In the ANME-D process, which was recently discovered, methane oxidation and denitrification occur under anaerobic conditions by a consortium of
Acknowledgments
O.M. was financially supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
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